Leishmania tarentolae: A new frontier in the epidemiology and control of the leishmaniases

Abstract Leishmaniasis (or the leishmaniases), classified as a neglected tropical parasitic disease, is found in parts of the tropics, subtropics and southern Europe. Leishmania parasites are transmitted by the bite of phlebotomine sand flies and million cases of human infection occur annually. Leishmania tarentolae has been historically considered a non‐pathogenic protozoan of reptiles, which has been studied mainly for its potential biotechnological applications. However, some strains of L. tarentolae appear to be transiently infective to mammals. In areas where leishmaniasis is endemic, recent molecular diagnostics and serological positivity to L. tarentolae in humans and dogs have spurred interest in the interactions between these mammalian hosts, reptiles and Leishmania infantum, the main aetiologic agent of human and canine leishmaniasis. In this review, we discuss the systematics and biology of L. tarentolae in the insect vectors and the vertebrate hosts and address questions about evolution of reptilian leishmaniae. Furthermore, we discuss the possible usefulness of L. tarentolae for new vaccination strategies.


THE STORY OF TWO SISTERS: REPTILIAN AND MAMMALIAN LEISHMANIAS
Trypanosomatids of the genus Leishmania (Kinetoplastida, Trypanosomatidae) are responsible for a significant health burden to mammals, including humans, in many tropical, subtropical and temperate regions, with 20 Leishmania spp. associated with human diseases (Okwor & Uzonna, 2016;Otranto & Dantas-Torres, 2013). For example, zoonotic visceral leishmaniasis caused by Leishmania infantum is a neglected disease of medical and veterinary importance worldwide, with the agent being transmitted by sand flies of the genera Phlebotomus in the Old World (Maroli et al., 2013) and Lutzomyia in the New World (Dantas-Torres et al., 2012).
A group of 21 less-studied leishmaniae, belonging to Sauroleishmania, is usually associated with sand flies of the genus Sergentomyia, which have long been considered to feed primarily on cold-blooded vertebrates (Akhoundi et al., 2016). Among them, Leishmania (subgenus Sauroleishmania) tarentolae was described from the gecko Tarentola mauritanica in Europe, North Africa and the Middle East .
Although it has long been considered non-pathogenic and specific to its reptilian hosts, some strains of L. tarentolae (e.g., the strain  were shown under laboratory conditions to cause transient infections in mammalian cells, differentiating into the amastigote stage, but not efficiently replicating within mammalian macrophages (Adler, 1962;Breton et al., 2005;Taylor et al., 2010). However, the unexpected detection of L. tarentolae in a mummy  and in human blood  triggered further investigations of the role of this trypanosomatid in the context of the leishmaniases and their control. Other members of the subgenus Sauroleishmania, such as Leishmania adleri, have also been associated with cutaneous leishmaniasis in humans (Coughlan et al., 2017;Manson-Bahr & Heisch, 1961), reflecting the understudied status of Sauroleishmania. Moreover, understanding the biology of L. tarentolae is highly relevant, given the myriad of applications in biotechnology due to (i) apparent absence of pathogenicity for humans and other mammals, (ii) easy and inexpensive cultivation and (iii) robustness as a platform for the production of recombinant proteins (Klatt et al., 2019;Niimi, 2012). For example, L. tarentolae exhibits mammalian-like post-translational modifications, which makes it a useful source for expressing functional mammalian antibody fragments and human glycoproteins (Jørgensen et al., 2014;Klatt & Konthur, 2012), such as N-glycans erythropoietin (Cantacessi et al., 2015) and amyloid precursor protein alpha (Klatt et al., 2013). Importantly, the finding of L. tarentolae in dogs, reptiles (i.e., both geckos and lizards), sand flies and humans in the same area where L. infantum is endemic (2021aIatta et al., 2021 opens many questions about the interactions between both trypanosomatid flagellates, potentially offering new opportunities for vaccines and/or immune-protection strategies to control canine and human leishmaniases. This review provides a comprehensive account of the main features of L. tarentolae systematics, phylogenetics and evolution, along with its biology in the insect vectors and the vertebrate hosts.

ORIGIN, EVOLUTION AND SYSTEMATICS OF SAURIAN-ASSOCIATED LEISHMANIA
The genus Trypanosoma has long been considered the most basal trypanosomatid branch, supporting the dixenous origin of this family.
However, the branching of the recently described monoxenous flagellate Paratrypanosoma confusum between free-living bodonids and parasitic trypanosomatids (Flegontov et al., 2013), favours the insectfirst scenario, in which the ancestral flagellate first invaded insects, and then only subsequently colonized vertebrate hosts, probably through blood feeding . The derived dixenous lifestyle evolved from the monoxenous one several times independently, initially in Trypanosoma and later on in the two-host genera Leishmania and Phytomonas, which are phylogenetically nested within the monoxenous trypanosomatids (Lukeš et al., 2014;Lukeš et al., 2014Lukeš et al., , 2018Maslov et al., 2013).
Despite the fact that Leishmania spp. have been intensively studied, there are many open questions regarding their taxonomy and phylogeny. Both concepts recently underwent substantial changes described below (Cupolillo et al., 2000;Espinosa et al., 2018;Harkins et al., 2016;Klatt et al., 2019;Kostygov & Yurchenko, 2017;Kostygov, et al., 2021). All Leishmania spp. belong to the subfamily Leishmaniinae within the family Trypanosomatidae in the order Trypanosomatida ( Figure 1). On closer examination, leishmaniae are grouped together with the newly described monoxenous genera Novymonas, Borovskyia and Zelonia in the infrafamily Leishmaniatae, while two established and species-rich monoxenous genera, Leptomonas and Crithidia, together with Lotmaria form the infrafamily Crithidiatae ( Figure 1). With monophyly well supported, all dixenous leishmaniae form two major sister lineages informal designated as sections or divisions: section Paraleishmania brings together the genera Endotrypanum and Porcisia (formerly Paraleishmania; see Kostygov & Yurchenko, 2017), while the genus Leishmania belongs to the section Euleishmania (the true Leishmania). Members of this genus are further divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia (formerly the Leishmania enriettii complex) (Figure 1). The subgenus Sauroleishmania was established half a century ago, although its type species L. tarentolae was described much earlier , and includes more than 20 species, which are restricted to the Old World (Akhoundi et al., 2016). Sauroleishmania spp. are known as reptilian parasites that have been consistently detected in various reptiles belonging to the saurian families Agamidae, Gekkonidae, Lacertidae, Scincidae and Varanidae originating from Mediterranean Europe, North Africa and the Middle East Wilson and Southgate, 1979), yet there are some interesting exceptions. Unlike most Sauroleishmania spp., L. adleri is capable of infecting mammals (Coughlan et al., 2017) and causes transient skin symptoms in humans (Manson-Bahr & Heisch, 1961) and asymptomatic infections in hamsters and mice (Adler, 1962). An undescribed species of Sauroleishmania (different from L. adleri and L. tarentolae) was found to cause visceral leishmaniasis in humans and dogs in China Yang et al., 2013).
Moreover, L. tarentolae promastigotes are capable of invading mammalian (including human) dendritic cells (DC) and macrophages, where they differentiate into an amastigote-like form, yet there is no unambiguous evidence of their replication (Breton et al., 2007;Taylor et al., 2010).
There are three mutually exclusive hypotheses postulating the origins of the genus Leishmania from the Palearctic or the Neotropics, or from the supercontinent before its split into present continents (Akhoundi et al., 2016;Harkins et al., 2016;Klatt et al., 2019;Lukeš et al., 2007;Schönian et al., 2018). The oldest fossil record of a protist parasite is represented by Paleoleishmania proterus found in the midgut lumen of a blood-filled female of the sand fly Palaemyia burmitis entrapped in mid-Cretaceous amber (∼100 MYA) in Myanmar (Poinar, 2004;Poinar & Poinar, 2004a). Promastigotes were mixed with nucleated reptilian blood cells, likely representing the ancestor of the genus Sauroleishmania (Poinar & Poinar, 2004a, 2004b. This finding implies that Sauroleishmania forms a sister clade to all other Leishmania species. However, the phylogenetic position of Sauroleishmania between the mammal-infecting subgenera Leishmania and Viannia suggests that this species-rich subgenus switched from mammals to reptiles (Klatt et al., 2019;Schönian et al., 2018). Although the available fossil record supports reptiles as early hosts of Leishmania-like parasites, the reptileinfecting subgenus Sauroleishmania must have arisen later, after the adaptation of Leishmania to mammals. While subsets of data can be used to support each of these hypotheses, the prevailing view places the origin of Leishmania in the Mesozoic, prior to the breakup of Gondwana.

GUT FEELING: LEISHMANIA TARENTOLAE DEVELOPMENT IN A SAND FLY GUT
Sauroleishmania spp. are generally transmitted by reptile-biting sand flies of the genus Sergentomyia, with many species found infected by various Sauroleishmania species (Karimi et al., 2014;Killick-Kendrick et al., 1986;Maroli et al., 1988;Rashti & Mohebali, 1994). Although Sergentomyia spp. feed primarily on reptiles, some species have been reported to bite mammals, including humans, raising a question about the role of these vectors in the transmission of mammal-infecting Leishmania species, particularly L. infantum (Maia & Depaquit, 2016).
Nevertheless, involvement of other sand fly genera in Sauroleishmania transmission should also be considered. Indeed, L. tarentolae DNA was recently detected in Phlebotomus perfiliewi , Phlebotomus perniciosus  and heavy late-stage infections were demonstrated experimentally in Phlebotomus papatasi (Adler & Theodor, 1929), P. perniciosus and Phlebotomus sergenti (Ticha et al., 2021). This may be due to the fact that many Phlebotomus species are opportunistic feeders, and their hostseeking behaviour may vary depending on the location, season and host availability (Quate, 1964). Their willingness to feed on cold-blooded animals has been repeatedly documented, with a prominent case of P.
papatasi (Adler & Theodor, 1929;Belova, 1971;Quate, 1964), which is susceptible to Sauroleishmania spp. infection (Adler & Theodor, 1929;Ticha et al., 2021). Collectively, these data suggest that sand flies of the genus Phlebotomus may play a role as alternative vectors in the circulation of L. tarentolae, and therefore in its transmission to non-reptilian hosts (Ticha et al., 2021). As Sergentomyia is a genus exclusively present Based on their development in vectors, Lainson and Shaw (1987) classified parasites of the genus Leishmania into three groups ( Figure 2(a)). The Suprapylaria (subgenus Leishmania) includes mammal-infecting species of the Old World (e.g., L. infantum), whose development is restricted to the midgut. The Peripylaria (subgenus Viannia) encompasses mammal-infecting species of the New World (e.g., Leishmania braziliensis), which develop in the hindgut and then migrate to anterior midgut. The third group consisting of reptileinfecting species (subgenus Sauroleishmania, including L. tarentolae) was named as Hypopylaria (Figure 2(a)). The development of these parasites was believed to be limited to the hindgut, suggesting that transmission to reptiles occurs when infected sand fly is ingested.
In contrast, species with suprapylarian or peripylarian type of development are transmitted to mammals by sand fly bites (Bates, 2007).
However, some Sauroleishmania species are capable of an anterior migration in the sand fly gut, associated with colonization of the anterior midgut (Figure 2(b)) (Adler & Theodor, 1929, 1935Ticha et al., 2021) and it is assumed that the hypopylarian type of development occurs only in some Sauroleishmania-sand fly combinations. The F I G U R E 2 Sand fly digestive tract and summary Suprapylaria/Peripylaria/Hypopylaria (a) and development of Leishmania tarentolae (b) development seems to be influenced by the insect, since L. tarentolae undergoes the peripylarian type of development in P. papatasi and P. perniciosus, yet the hypopylarian development prevails in P. sergenti (Ticha et al., 2021). Such variability in the vector-parasite interaction may be due to different behaviour of Sauroleishmania species to escape from the blood meal surrounded by the peritrophic matrix and by their capacity to attach to different parts of the sand fly gut.
It has also been proposed that the hypopylarian type of development occurs when promastigotes cannot cross the peritrophic matrix and are passed into the hindgut (Bates, 2007). Indeed, the role of peritrophic matrix in parasite life cycle is important. For example, its delayed degradation in Sergentomyia schwetzi is known to cause the refractoriness of this vector to mammal-infecting Leishmania species (Sádlová et al., 2018). However, further studies on Sauroleishmaniasand fly interactions are necessary to confirm these hypotheses.
Although S. minuta, the proven natural vector of L. tarentolae, is one of the most abundant sand flies in the Mediterranean (Maroli et al., 1988), only two studies described the development of L. tarentolae in this sand fly species (Adler & Theodor, 1935;).
Females of S. minuta (erroneously referred to as Phlebotomus parroti in the original description  were experimentally infected by feeding on gecko T. mauritanica carrying a mixed infection of L. tarentolae and Trypanosoma platydactyli. Both parasites acquired an anterior position in the sand fly gut, with Sauroleishmania promastigotes found in the midgut and cardia, but not in the hindgut (Adler & Theodor, 1935). Recently, the development of L. tarentolae in Malpighian tubules of three Phlebotomus species was experimentally demonstrated (Ticha et al., 2021). The localization in Malpighian tubules is rather unique for the genus Leishmania, with only two other reports of unidentified promastigotes in Sergentomyia garnhami, Sergentomyia antennata (Kaddu, 1986) and in S. minuta (Killick-Kendrick et al., 1979). An examination of laboratory bred S. minuta females that were allowed to feed on naturally infected geckos revealed that L. tar-entolae is able to colonize the Malpighian tubules of both Sergentomyia spp. and Phlebotomus spp. (Ticha et al., unpublished). Though there are only few records of Sauroleishmania morphological forms in vectors (Adler & Theodor, 1929, 1935, they do not differ from those known for Leishmania in mammals, but the infectious stages for reptiles are not known (Bates, 2007). So far, a successful experimental transmission of Sauroleishmania from sand flies to reptilian hosts has not been demonstrated.
Two possible modes of transmission may be considered. The hypopylarian type of development of some Sauroleishmania species suggests that reptiles become infected by ingestion of a sand fly. On the contrary, species with the peripylarian type might be transmitted by bite, via the pool-blood feeding mechanism, similarly to mammalinfecting Leishmania (Bates, 2007). Colonization of the stomodeal valve and disruption of its surface are essential for effective transmission of Leishmania to its mammalian hosts, as it facilitates the regurgitation of parasites from the midgut (Dostálová & Volf, 2012). The presence of L. tarentolae promastigotes in the cardia and colonization of the stomodeal valve in Phlebotomus spp. (Adler & Theodor, 1929;1935) support the idea of transmission by bite (Figure 3). However, the localization of L. tarentolae in Malpighian tubules raises a third possible scenario, namely the transmission by prediuresis. When feeding on a host, sand fly females regularly excrete urine to concentrate proteins in bloodmeal and restore weight and water balance (Sádlová et al., 2013). Viable L. major promastigotes, including the metacyclic form, were found in urine droplets discharged by infected P. papatasi and Phlebotomus duboscqi females, during feeding (Sádlová & Volf, 1999).
Leishmania promastigotes in urine droplets may enter bite wounds or mucosal membranes. As urine is secreted from Malpighian tubules and passes the hindgut (both tissues being the typical location of L. tarentolae promastigotes), the role of prediuresis in Sauroleishmania transmission should be considered plausible and therefore further studied.

F I G U R E 3
Life cycle of Leishmania tarentolae in vectors and hosts. In reptiles, amastigote-like forms develop in blood cells, and parasite DNA has been detected in blood and erythropoietic organs. Sand flies ingest infected blood cells and parasites differentiate into promastigote forms and undergo hypopylarian or peripylarian type of development. Possible transmission routes to vertebrate hosts are via sand fly bite, oral ingestion of the fly or contaminative way by prediuresis. Transmission and development in mammals are not known

MAMMALIAN EXPOSURE TO LEISHMANIA TARENTOLAE AND THE ROLE OF REPTILES IN THE LEISHMANIASES
In the early years, L. tarentolae was classified as Leptomonas while researchers investigated T. mauritanica gecko as a possible reservoir of a zoonotic disease called Biskra boil and caused by Leishmania spp. (Sergent et al., 1914). Soon after, while describing different types of reptilian flagellates from Egypt, Wenyon  mentioned that the species isolated by Sergent (Sergent et al., 1914) was in fact a Leishmania, later becoming L. tarentolae. At the moment of the first isolation of L. tarentolae, some authors hypothesized that geckos could be reservoirs of cutaneous leishmaniasis caused by Leishmania tropica and/or Leishmania major (Chatton & Blanc, 1914;McMillan, 1966;Sergent et al., 1914;. Also, other Sauroleishmania species were suspected to be causative agents of cutaneous leishmaniasis or oriental sore. For example, L. adleri was isolated from the blood of Latastia longicaudata lizards in Kenya (Heisch, 1958), and was believed to be a strain of Leishmania donovani. Unlike L. tarentolae, more studies confirmed the pathogenic effect of L. adleri as the causative agent of cutaneous leishmaniasis in rodents and humans (Coughlan et al., 2017;Manson-Bahr & Heisch, 1961). It was hypothesized that interactions between mammalian and reptilian leishmania (i.e., L. tarentolae in mammals and L. donovani in reptiles) could ultimately result in partial dilution of species, thus immunization and protection, within the two sister clades (Mutinga & Ngoka, 1981).
Furthermore, additional attempts were made to identify and isolate Sauroleishmania from endemic areas of human and canine leishmaniasis. Axenic cultures of L. tarentolae were obtained from France (Gao et al., 2001) and Italy  with reports of L. tarentolae in different species of reptiles (Klatt et al., 2013;Klatt et al., 2019; (Elwasila, 1988;Klatt et al., 2013;. While studying the molecular prevalence of L. infantum in human donors, sand flies and dogs from central Italy, L. tarentolae was detected by nested-PCR in humans and sand flies (i.e., Phlebotomus and Sergentomyia) . This finding was most likely related to the Sergentomyia spp. transmitting L. tarentolae while feeding on humans . Moreover, the substantial reduction in anti-L. infantum antibody titres of more F I G U R E 4 Distribution map of Leishmania tarentolae based on isolates and molecular detection in reptiles, sand flies and mammals (Annex 1). Dark green represents confirmed distribution; question mark refers a controversial finding concerns parasites detected in bone marrow and intestinal tissue samples from a 300-year-old Brazilian mummy based on a kDNA amplicon matching to L. tarentolae , which, however, does not agree with the geographical distribution of the subgenus Sauroleishmania than half of the population of L. infantum-seropositive and clinically healthy sheltered dogs, sampled throughout the year (that is, during the transmission and non-transmission season), raised questions about the possibility of dogs being exposed to L. tarentolae (Cavalera et al., 2021). In fact, circumstantial evidence suggested by the seasonal varia- Capability of pathogenic mammalian-associated Leishmania to infect reptiles was studied in the late 1960s and 1970s and was ultimately disregarded, mainly given the physiological differences between mammals and reptiles (e.g., reptiles being ectotherms and mammals endotherms) (Belova, 1971;McMillan, 1966). Nevertheless, Belova

LEISHMANIA TARENTOLAE AND THE CELLULAR MODEL
Leishmania tarentolae is broadly used for a range of biotechnological applications, from protein production to its exploitation as a model for drug discovery (Klatt et al., 2019). In the area of bio-molecular studies, L. tarentolae was firstly exploited to investigate gene amplification (Ouellette et al., 1991;White et al., 1988) and RNA editing in the mitochondrion (Blum et al., 1990). In parallel, L. tarentolae was developed as a platform for recombinant protein production (Cantacessi et al., 2015), and then commercialized by Jena Bioscience (Jena, Germany) under the name LEXSY. The LEXSY system allows the expression of target proteins either in a constitutive or inducible form, as intracellular or secretory molecules (https://www.jenabioscience.com/). The strain P10, on which the LEXSY system is based, was likely derived from the TARII/UC strain of the parasite, isolated by Parrot from an Algerian gecko (T. mauritanica) (Klatt et al., 2019;Parrot, 1949). Among the variety of microbial and cellular platforms to produce recombinant proteins (e.g., prokaryotes, yeasts, mammalian cells, insect cells), L. tarentolae found its niche thanks to some specific characteristics. First, the maintenance and growth of L. tarentolae is accomplished at a low cost: promastigotes are easily cultured in aerobic conditions as continuous suspension culture at 26 • C, in different synthetic media (Cantacessi et al., 2015;Kushnir et al., 2005). Second, growth characteristics are suitable to scale the production to industrial levels, by growing parasites in bioreactors, with the potential of harvesting high yields of recombinant proteins from engineered strains (Niimi, 2012). Third, L.
tarentolae presents a protein glycosylation pattern that is very likely to overlap that of pathogenic Trypanosomatidae (Murphy et al., 2020), but is also similar to that of mammals (Cantacessi et al., 2015). Based on the above characteristics, L. tarentolae is an interesting system for protein studies (e.g., X-ray Crystallography) and for the production of protein antigens, for example for sero-diagnostic applications and vaccine development. To date, the use of this protist to produce antigens for diagnostic application has been limited to experimental studies on antigens from pathogenic species of Leishmania or Trypanosoma (Rezaei et al., 2019;Rooney et al., 2015) and from viruses (Baechlein et al., 2013;Varotto-Boccazzi et al., 2021). In this context, a recent paper showed that a recombinant protein produced in L. tarentolae allows reliable serological diagnosis of SARS-CoV-2 infection (Varotto-Boccazzi et al., 2021). However, while in the presence of biantennary glycosylation structures, N-glycosylation in L. tarentolae is not completely overlapping that of mammals (Cantacessi et al., 2015). Therefore, the capability of a given viral antigen produced in L. tarentolae to match the diagnostic patterns should always be carefully compared with the same antigen expressed from mammalian cells.
In view of its safety and easy culturing, L. tarentolae has been investigated as a surrogate pathogen in candidate vaccines, aimed at protecting against human pathogenic Leishmania species. In a first seminal paper, Breton et al. (2005) showed that L. tarentolae promastigotes are engulfed by DCs in vitro, inducing proper maturation of these cells, with expression of major histocompatibility complex class II (MHCII) and costimulatory molecules. More importantly, this study showed that intraperitoneal administration of live L. tarentolae in BALB/c mice determined polarization of the immune response toward Th1 pathway, with significant protection against challenge with L. donovani. In successive pre-clinical studies, live L. tarentolae promastigotes were assayed as candidate vaccines in association with adjuvants, with cross-protective immunity against L. major (Haghdoust et al., 2022;Keshavarzian et al., 2020). While the above studies had been performed using non-engineered strains of L. tarentolae, thus exploiting some form of cross-immunity with human pathogenic species, other studies employed genetically modified strains of L. tarentolae, engineered for expression of antigens from human pathogenic leishmanias (Salari et al., 2020;Saljoughian et al., 2013) and/or of immune-modulating molecules (Montakhab-Yeganeh et al., 2017), such as proteins from the sand fly saliva (Katebi et al., 2015). These stud-ies generally showed that whole live promastigotes from engineered strains of L. tarentolae determined protection in animal models against pathogenic species, including L. infantum and L. major.
In parallel with the above studies on anti-Leishmania vaccines, L. tarentolae was investigated for its potential as a platform to generate anti-viral vaccines. Targeted viruses include human immunodeficiency virus 1 (Breton et al., 2007), human papillomavirus (Bolhassani et al., 2015) and hepatitis C virus (Ansari et al., 2019). The engineered strains of L. tarentolae have so far been assayed only in animal models, either as living vehicles for the antigens (Ansari et al., 2019;Breton et al., 2007), or just as biofactories for antigen production (Bolhassani et al., 2015). The first approach is obviously based on the assumption that the targeting of L. tarentolae to DCs should facilitate the delivery of viral antigens to secondary lymphoid organs, ensuring their presentation to CD4+T cells (Breton et al., 2005;Breton et al., 2007). In the second approach, the antigen is administered after purification. Overall, studies above led to encouraging results in animal models, in terms of the generation of virus-neutralizing antibodies.

CONCLUDING REMARKS
Leishmania tarentolae is a promising protist for its biotechnological applications, of which very little is known regarding its biological cycle, transmission pathways and overall biology. However, the interaction Finally, although L. tarentolae has historically been considered nonpathogenic and unlikely to infect mammals, some cultured strains have been shown to be transiently infectious to mammals. The fact that reference laboratory strains are probably non-infectious even for reptiles, spurs the need for new isolates to fully understand the natural development of L. tarentolae in reptiles and in mammals. In addition, this species has been studied as a model for anti-Leishmania vaccines and a platform to generate antiviral vaccines with overall encouraging results in animal models, in terms of the generation of virus-neutralizing antibodies.
The overall picture presented in this review is useful in understanding the implications of the interactions of these sister clades Leishmania, which may be applied knowledge to improve diagnostic tools, efficient control and treatment of a neglected disease that is a high burden to our society.